EP1465657A2 - Methode permettant de renforcer l'immunogenicite par liaison covalente d'antigenes a des proteines a la surface de cellules dendritiques - Google Patents

Methode permettant de renforcer l'immunogenicite par liaison covalente d'antigenes a des proteines a la surface de cellules dendritiques

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Publication number
EP1465657A2
EP1465657A2 EP02796072A EP02796072A EP1465657A2 EP 1465657 A2 EP1465657 A2 EP 1465657A2 EP 02796072 A EP02796072 A EP 02796072A EP 02796072 A EP02796072 A EP 02796072A EP 1465657 A2 EP1465657 A2 EP 1465657A2
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Prior art keywords
antigen
cells
dcs
dendritic cells
tumor
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German (de)
English (en)
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EP1465657A4 (fr
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Richard B. Bankert
Thomas F. Conway
Nejat Egilmez
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Health Research Inc
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Health Research Inc
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0639—Dendritic cells, e.g. Langherhans cells in the epidermis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/385—Haptens or antigens, bound to carriers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/10—Cellular immunotherapy characterised by the cell type used
    • A61K40/19—Dendritic cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/20—Cellular immunotherapy characterised by the effect or the function of the cells
    • A61K40/24—Antigen-presenting cells [APC]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/41—Vertebrate antigens
    • A61K40/42—Cancer antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/41—Vertebrate antigens
    • A61K40/42—Cancer antigens
    • A61K40/4244—Enzymes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/45—Bacterial antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6901—Conjugates being cells, cell fragments, viruses, ghosts, red blood cells or viral vectors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
    • A61K2039/6031—Proteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/62—Medicinal preparations containing antigens or antibodies characterised by the link between antigen and carrier
    • A61K2039/627—Medicinal preparations containing antigens or antibodies characterised by the link between antigen and carrier characterised by the linker
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K40/00
    • A61K2239/46—Indexing codes associated with cellular immunotherapy of group A61K40/00 characterised by the cancer treated
    • A61K2239/50—Colon
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/05—Adjuvants
    • C12N2501/056—Immunostimulating oligonucleotides, e.g. CpG

Definitions

  • the present invention relates to the general field of immunotherapy and more particularly provides a method for increasing immunogenicity of an antigen.
  • DCs Dendritic cells
  • APCs antigen presenting cells
  • DCs are located throughout the body and are most highly concentrated at the organism's interface with the environment (i.e. epidermis and dermis and the mucosal surfaces of the lung and gastrointestinal tract). It is here that DCs are considered to survey their surroundings and through the production of soluble factors, alert innate effectors to invasion by pathogens. Upon interaction with microbial products or inflammatory cytokines, DCs produce chemokines and cytokines that recruit and activate additional APCs and immune effector cells.
  • DCs In addition to mobilizing the innate response, DCs collect information from their microenvironment and serve as liaisons between the peripheral tissues and the na ⁇ ve T cells, which are limited to passage through blood and lymphoid organs.
  • the DCs, carrying antigen (Ag) encounter potentially receptive T cells in peripheral lymphoid tissues.
  • DCs have the ability to direct an effective adaptive immune response.
  • DCs deliver information in the form of foreign peptide bound to MHC molecules in concert with costimulatory molecules and soluble cytokine release to prime and direct the generation of a T cell response. Through their ability to direct the initiation of both the innate and adaptive immune responses DCs play an invaluable role in immune defense.
  • a system has developed in DCs which directs a host organism's response toward cellular (T H I) or humoral (T H 2) immunity.
  • DCs convey information about their sentinel experience in the form of three instructive signals.
  • the first signal advises of foreignness of a peptide in the context of MHC
  • the second signal conveys the presence or absence of danger through the expression of costimulatory molecules.
  • the third signal supplies information (gained through evolution) that directs the type of immune response that should be mounted.
  • Bacterial DNA contains motifs that include unmethylated Cytidine- phosphate-Guanosine (CpG) repeats.
  • CpG Cytidine- phosphate-Guanosine
  • the CpG repeats and their specific flanking sequences impart a potentiating activity to the bacterial DNA (Kreig et al. 2000).
  • the immunological effects of bacterial CpG containing oligonucleotides are profound. Unmethylated bacterial CpG repeats bind to a receptor, toll like receptor-9 (TLR-9) on DCs and induces maturation of immature cells.
  • TLR-9 toll like receptor-9
  • CpG oligonucleotides induce the third signal by causing DC production of IL-12 (Sparwasser 1998, Brunner et al. 2000).
  • IL-12 is one of the most potent factors in the induction of a cellular or THI response (Manetti 1993).
  • a T H I response results in the development of cells that produce high amounts of IFN ⁇ (Moser and Murphy 2000).
  • a T H 1 type response has also been effective in experimental anti-tumor immune responses (Mosmann T 1996, Trincheri 1994, Brunner et al 2000).
  • DCs have been used extensively for vaccination against a variety of protein antigens and DC vaccines have also been found to be powerful stimulators of the cellular immune response to tumors (reviewed in Banchereau et al. 2000).
  • DCs act as potent initiators of tumor immunity in murine tumor models.
  • the development of tumors in experimental animals can be induced by injection of established tumor cell lines derived from a number of different tissues (Brunner et al. 2000).
  • DCs pulsed with tumor antigen or cell lysate are effective anti-tumor vaccines against subsequent tumor cell challenge.
  • a number of tumor antigen pulsing methods have met with success in experimental settings.
  • Co-culture (Celluzi 1998) or fusion of DCs with whole tumor cells (Gong 1997) provides protection from subsequent challenge with viable tumor cells.
  • CT26 is an N-nitroso-N-methylurethane induced BALB/c undifferentiated colon carcinoma. This tumor grows progressively in animals after subcutaneous or intra- venous injection (Wang et al. 1995). The transfection of this tumor with the bacterial lac-Z gene leads to the expression of ⁇ -galactosidase in the tumor cells.
  • CT26.CL25 This variant of CT26, that is called CT26.CL25 has been established as a progressively growing tumor (Wang et al. 1995).
  • ⁇ -galactosidase acts as a surrogate tumor antigen.
  • ⁇ -Galactosidase ( ⁇ -gal) is an enzyme that cleaves substrates including lactose and o-nitrophenyl- ⁇ -D-galactopyranoside (ONPG).
  • ⁇ -Gal has been extensively studied in investigations into the E. coli lactose operon and it has also been used as a marker for measuring the efficiency of gene transfer. As a result of these past studies, many assays have been developed to detect and quantify the presence and activity of ⁇ -gal.
  • Each of the referenced techniques are associated with either significant technical expertise (cationic liposome association and cationic fusogenic peptides) or labor intensive isolation of antigen and cell culture techniques (purification of apoptotic bodies). These methods may also require construction and expression of fusion proteins, the availability of a specific antibody-antigen pair, complex manipulations or a large amount of tumor material to enhance targeting to antigen presenting cells. These drawbacks may limit the utility of such techniques.
  • the present invention provides compositions and methods for enhancing the immunogenicity of antigens.
  • the method comprises covalently linking (also referred to as covalent coupling) the antigen to proteins or glycoproteins on the surface of dendritic cells and using the dendritic cells to elicit an immune response.
  • the mild biochemical modification employed by this approach minimizes denaturation of the Ag.
  • the viability of cells is preserved.
  • a model tumor antigen, ⁇ -galactosidase ( ⁇ -gal) was covalently coupled to proteins or glycoproteins on the surface of DCs.
  • DCs with covalently linked Ag on their surface were compared to DCs pulsed with soluble Ag for the ability to generate a tumor specific immune response in mice.
  • Covalently linked ⁇ -gal-DCs proved to be superior to soluble ⁇ -gal loaded DCs in generating both protective and therapeutic anti-tumor immunity.
  • This technique can be used with a wide range of antigens such as proteins or peptide fragments of known tumor or microbial proteins or tumor cell and bacterial lysates that contain a variety of antigenic components.
  • the invention also provides compositions for eliciting an immune response.
  • the composition comprises dendritic ells having one or more antigens covalently linked to the surface molecules, preferably proteins or glycoproteins.
  • the invention also provides a method for making a composition for eliciting an immune response in an individual.
  • the method comprises obtaining dendritic cells from the individual (or a syngeneic source), covalently coupling an antigen to one or more proteins or glycoproteins on the surface of the dendritic cells; and reinfusing the covalently coupled dendritic cells into the individual.
  • the invention also provides a method of reducing the growth of a tumor by obtaining a tissue sample from the tumor; isolating an antigen from the tissue sample or preparing cell lysates; obtaining dendritic cells from the individual; covalently linking the purified, partially purified or unpurified antigen to one or more surface proteins on the dendritic cells; and reinfusing the covalently linked dendritic cells into the individual.
  • the invention also provides a method for reducing the recurrence of the growth of tumors in an individual in which the tumor has been surgically removed by obtaining a tissue sample from the tumor which has been surgically removed; isolating an antigen from the tissue sample or preparing a cell lysate from the tumor; obtaining dendritic cells from the individual; covalently linking the antigen or the cell lysate to one or more surface proteins or glycoproteins on the dendritic cells; and reinfusing the covalently linked dendritic cells into the individual.
  • the invention also provides a method for reducing the incidence of occurrence of tumors by identifying an antigen known to be present in the tumors, obtaining dendritic cells from an individual; covalently linking the antigen from the tumor to one or more surface proteins on the dendritic cells; and reinfusing the covalently linked dendritic cells into the individual.
  • DC2 human lymphoid DC subset DMEM, Dulbecco's Modified Eagle Medium
  • TFN ⁇ Interferon gamma Ig, hnmunoglobulin
  • IL-_ Interleukin-_ ip
  • intraperitoneal iv intravenous LPS
  • MLR Mixed Lymphocyte Reaction or Mixed Leukocyte Reaction
  • RANK Receptor Activator of Nuclear factor-kappaB rmGM-CSF, recombinant mouse Granulocye, Macrophage Colony Stimulating Factor
  • SCID Severeve Combined Immunodeficient (mouse) SLC, Secondary lymphoid tissue chemokine
  • T H I T cell helper subset 1 (cellular immune response)
  • T H 2 T cell helper subset 2 (humoral immune response)
  • TNF ⁇ Tumor Necrosis Factor-alpha TRANCE
  • TNF tumor necrosis factor
  • Day 10 bone marrow culture derived cells exhibit dendritic cell morphology as exemplified by extensive cytoplasmic "veils" in non-adherent cells and stellate projections in attached cells.
  • Figure 2 Flow cytometric analysis of day 7 GM-CSF cultured bone marrow dendritic cell phenotype.
  • FIG. 3 Flow cytometric investigation of DC maturation.
  • Day 10 BM cultured cells were assayed for MHC class II (y axis) and CD86 (x axis) expression 16 hours after transfer to a 6 well tissue culture plate.
  • the cells were incubated overnight with media + GMCSF only (a) or with the addition of lOOng/ml LPS (b), 1 ⁇ g/ml CpG 1826 (c), or 6 ⁇ g/ml CpG 1826 (d).
  • FIG. 4 Stimulation of the MLR by GM-CSF cultured BM cells. 5 x 10 5 Allogeneic T cells were mixed with varying doses of day 9 GM-CSF cultured BM cells ( ⁇ ) or splenoctyes ( ⁇ ). After a three-day incubation period, cell number/activity was determined by an MTT assay. Each data point represents three separate wells. The error bars are demonstrative of one standard deviation.
  • FIG. 5 IL-12 production by GM-CSF BM cells in response to CpG 1826.
  • Day 9 GM-CSF cultured bone marrow cells were transferred to a tissue culture treated well at 5xl0 5 cells in 0.5 ml complete medium per well.
  • To each well was added 0.5 ml of media with no additional factors (None) or a maturation factor: LPS (lOOng/ml final cone), or CpG 1826 at a final concentration of 1, 6, or 12 ⁇ g/ml.
  • Medium supernatant was collected and assayed 20h later.
  • Each column is representative of triplicate samples from duplicate plates. The error bars are representative of one standard deviation.
  • Figure 6. Titration of the optimal dose of CpG for the induction of IL-12 by
  • BM cultured cells Day 9 GM-CSF cultured bone marrow cells were transferred to a tissue culture treated well at 5xl0 5 cells in 0.5 ml complete medium per well. To each well was added 0.5 ml of media with a titrated dose of CpG 1826 at final concentrations as labeled. Medium supernatant was collected and assayed 20h later. Each column is representative of triplicate readings of duplicate wells. The error bars are representative of one standard deviation.
  • Figure 7 Coupling of a protein antigen to proteins on the surface of DCs through the use of SPDP.
  • SPDP a 3-(2-pyridyldithio) propionyl (PDTP) groups into a protein by aminolysis.
  • PDTP 3-(2-pyridyldithio) propionyl
  • FIG. 8 PDTP- ⁇ -Gal binding to the surface of DCs. Either soluble ⁇ -Gal (open bar) or PDTP modified ⁇ -Gal (colored bar) was added to a titrated number of DCs. The cells were washed extensively after a 1-hour incubation. Cell surface ⁇ - Gal activity was determined by an ELIS A to detect the cleavage of ONPG. Each column is representative of triplicate wells. The error bars are representative of one standard deviation. This experiment was repeated 3 times with similar results.
  • FIG. 9 Loss of ⁇ -Gal activity through the use of MESNa.
  • lxlO 6 DCs were incubated with PDTP- ⁇ -Gal for 60 min. followed by treatment with MESNa (open bar) or PBS (filled bar).
  • the cells were washed extensively after a 1-hour incubation and ⁇ -Gal activity was determined using an ELIS A.
  • Each column is representative of triplicate wells. The error bars are demonstrative of one standard deviation. This experiment was repeated twice with similar results.
  • FIG. 10 Internalization of covalently coupled surface ⁇ -Gal by DCs. Initially, 1.2xl0 7 DCs were incubated with PDTP- ⁇ -Gal for 60 min. DCs were incubated at 37°C for the indicated amount of time. At the end of the incubation period the DC were treated with MESNa. Intact (open bar) or lysed (closed bar) cells were then assayed for ⁇ -Gal activity using an ELIS A with lxlO 6 DCs per well. Each column is representative of triplicate wells. The error bars are one standard deviation. This graph is representative of three experiments. Figure 11. Ag pulsed DCs stimulate the allogeneic MLR.
  • Escalating doses of unpulsed DC ( ⁇ ), soluble ⁇ -Gal (D) and PDTP- ⁇ -Gal ( • ) were added to 2x10 5 allogeneic lymphocytes. After a three-day incubation period, cell number/activity was determined by an MTT assay. Each data point represents three separate wells. The error bars are demonstrative of one standard deviation from the mean.
  • FIG. 12 Survival of DCs vaccinated mice after CT26.CL25 challenge.
  • Groups of mice were vaccinated with 5xl0 5 cells; unpulsed DC (O), soluble ⁇ -Gal pulsed DCs ( ⁇ ), PDTP- ⁇ -Gal pulsed DCs (0) or left unvaccinated (A).
  • Mice were challenged subcutaneously with 5xl0 5 CT26.WT tumor cells 17 to 49 days after treatment. Tumor growth was monitored weekly and survival ended at sacrifice, when one dimension of the tumor exceeded 2cm. The data from 6 experiments were combined to produce this figure.
  • DCs A
  • PDTP- ⁇ -Gal pulsed DCs •
  • left unvaccinated ⁇
  • mice Twenty days later the mice were subcutaneously challenged with 5x105 CT26.WT tumor cells. Each data point represents the average tumor size in five mice. Tumor growth was monitored weekly. Tumor volume was calculated as described. Mice were sacrificed when one dimension of their tumor exceeded 2cm. This preparation of DCs (PDTP- beta-Gal pulsed) was able to protect mice against challenge with CT26.CL25 ( ⁇ ). There was no significant difference in tumor growth between any of the cell lines that were challenged with CT26.WT (p>0.5).
  • FIG. 14 Survival of mice challenged with CT26.CL25 7 weeks after DC vaccination. Groups of mice were vaccinated with 5xl0 5 cells; unpulsed DCs ( ⁇ ), soluble ⁇ -Gal pulsed DCs (A), or PDTP- ⁇ -Gal pulsed DCs (•). Forty-nine days later the mice were subcutaneously challenged with 5x105 CT26.CL25 tumor cells. Tumor growth was monitored weekly and survival ended at sacrifice, when one dimension of the tumor exceeded 2cm. The data from 2 experiments were combined to produce this figure.
  • FIG. 15A-C DC based treatment of established tumors. Mice were injected S.C. with 5x10 5 CT26.CL25. Ten days later the mice were treated with a contra lateral S.C. DC injection as indicated. Each group contained 5 mice and each mouse is represented individually. Tumor growth was monitored weekly. Tumor volume was calculated as described. Mice were sacrificed when one dimension of their tumor exceeded 2cm.
  • FIG. 16 The addition of CpG to in vitro DC cultures results in an increase in antigen specific IFN ⁇ producing cells.
  • Mice were vaccinated as indicated on the X-axis. Twelve days later splenocytes from the vaccinated animals were isolated and restimulated for 6 days with ⁇ -gal positive P13.4 tumor cells. The cells were recovered and incubated overnight in an ELISPOT plate in the presence of PI 3.4 cells. The responder cells were titrated at 2xl0 5 (gray column), lxlO 5 (white column) and 5x10 4 (black column) per well. The plates were used in an ELISPOT assay to detect IFN ⁇ producing cells. Each column is the average of triplicate wells. The error bars are one standard deviation from the mean. This experiment was repeated and yielded comparable results.
  • FIG. 1 IFN ⁇ producing cells obtained from ⁇ -gal-pulsed and ⁇ -gal- conjugated vaccination. Mice were vaccinated as indicated on the X-axis. Splenocytes from the vaccinated animals were isolated and restimulated with irradiated P13.4 cells. Five days later the cells were recovered and incubated overnight in an ELISPOT plate in the presence of ⁇ -gal positive PI 3.4 tumor cells. The responder cells were assayed at 2xl0 5 (gray column), and lxlO 5 (white column). The plates were used in an ELISPOT assay to detect IFN ⁇ producing cells. Each column is the average of quadruplicate wells. The error bars are one standard deviation from the mean. This figure is representative of 3 independent experiments.
  • FIG. 1 TFN ⁇ production by CD8 + and CD8 " cells.
  • Mice were vaccinated as indicated on the X-axis. Splenocytes from the vaccinated animals were isolated and restimulated with irradiated PI 3.4 tumor cells for 5 days. The recovered cells were separated into CD8 + and CD8 " cells and then were incubated overnight in an ELISPOT plate in the presence of PI 3.4 cells. The responder cells were plated at 5xl0 4 CD8 + cells (gray column), and 5xl0 4 CD8 " cells (white column) in an ELISPOT assay to detect IFN ⁇ producing cells. Each column is the average of quadruplicate wells. The error bars are one standard deviation from the mean. This figure is representative of 2 independent experiments.
  • FIG. 19 Requirement of ⁇ -gal expressing cell line for in vitro restimulation period.
  • Mice were vaccinated as indicated on the X-axis. Splenocytes from the vaccinated animals were isolated and restimulated for 6 days with either ⁇ - gal negative CT26.WT cells (open columns) or with the ⁇ -gal expressing CT26.CL25 tumor cell line (filled columns). The recovered cells were then were incubated overnight in an ELISPOT plate in the presence of CT26.CL25 cells. The responder cells were plated in 3 wells and the average values are reported. The error bars are one standard deviation. This experiment was repeated twice at lower responder to stimulator cell ratios with similar results.
  • the present invention provides a method for enhancing the immunogenicity of antigens by covalently linking them to the proteins or glycoproteins on the surface of dendritic cells. Covalent linkage can be achieved by methods well known in the art. Once the antigen has been covalently linked to the DC surface, the DCs can then be used to elicit an immune response. As an illustration, dendridic cells from the bone marrow have been used.
  • the method of the present invention can be used with any antigen and can be used for prophylactic as well as therapeutic purposes.
  • antigens include but are not limited to tumor related antigens such as Immunoglobulin idiotypes, Mage, BAGE, MART, SV40T antigen, EBNA-1, Her-2/neu, Bcr/Abl, Ras, Tyrosinase, Alpha-fetoprotein, Prostate specific antigen; viral antigens such as viral coat proteins and viral capsid proteins; and bacterial antigens such as bacterial coat proteins and bacterial products such as heat killed toxins (e.g., tetanus toxoids) etc.
  • Antigens useful for the invention can be obtained commercially or prepared by standard methods.
  • tumor antigens can be obtained by preparation of tumor cell lysates which are prepared by repeatedly freezing and thawing tumor cells/tissues in phosphate buffered saline containing leupeptin and aprotinin (obtained from either fresh tumor biopsy tissues or from tumor cells generated in vitro by tissue culture). The freezing and thawing results in the lysis of cells. The tumor lysate is obtained by centrifugation and harvesting the supernatant fluid. The tumor cell lysates can be used immediately or frozen and stored at -70°C until ready for use. The cell lysate can itself be used for covalent coupling to DCs.
  • the antigen can be used in a purified form or in partially purified or unpurified form as cell lysate.
  • the experiments described here demonstrate that covalent linkage of Ag to the surface proteins or glycoproteins of DCs enhances the immune response elicited to that Ag.
  • the process of covalently coupling Ag to DCs is simple and mild enough that DC viability is well preserved. While not intending to be bound by any particular theory, it is considered that covalently linked antigen is internalized, processed and presented by DCs. This is evidenced by the vaccination of mice with PDTP- ⁇ -gal pulsed DCs and the subsequent recovery of CD8 + T cells that respond specifically to ⁇ -gal expressing tumor cells.
  • Covalent coupling of Ag to proteins or glycoproteins on the DC cell surface does not require complex biochemical or molecular manipulation.
  • Covalent coupling of antigen to proteins on the surface of DCs requires an initial mild modification of the antigen, followed by interaction with the DCs to effect a covalent association with the target cell. The reactions required for this procedure are performed under gentle physiological conditions, thereby minimizing denaturation of the antigen and preserving cell viability.
  • Covalent coupling of antigens to the surface proteins or glycoproteins of dendritic cells can be accomplished by well known methods that are within the purview of those skilled in the art.
  • a wide variety of compounds including homobifunctional and heterobifunctional reagents are available for covalent coupling.
  • heterobifunctional reagents allows for the covalent linkage of two proteins to each other through the use of two different reactive groups. Any reactive group can be used.
  • An example of such a bifuctional reagent is N-succinimidyl 3-(2-pyridyldithio) propionate (SPDP).
  • SPDP is a heterobifunctional reagent that works to covalently link together two proteins through the creation of a disulfide bond.
  • a .heterobifunctional reagent such as SPDP
  • coupling of the reagent to the first protein and linking it to the second protein can be carried out in separate sequential steps because the two reactive groups of the SPDP are directed toward different functional groups on the proteins.
  • SPDP acts primarily in the following way. SPDP contains one N-hydroxysuccinimide ester moiety and one 2-pyridyl disulfide moiety. The first reaction occurs when the hydroxysuccinimide esters react with the amino groups on the protein Ag, this reaction gives rise to stable amide bonds. Treatment of the protein Ag with iodoacetamide prior to reaction with SPDP blocks any free thiol groups that would react with the 2-pyridyldithio moiety of the SPDP molecule and allows the reaction to occur without intramolecular crosslinking or homoconjugation of the protein Ag.
  • the protein Ag now contains 3 -(2-pyridyldithio) propionyl (PDTP) groups that are able to react with a second protein.
  • PDTP 3-(2-pyridyldithio) propionyl
  • the 2-pyridyl disulfide groups then react with free thiol groups to form disulfide bonds.
  • heterobifunctional covalent linkers can also be used.
  • the coupling reaction is preferably mild for use in antigen conjugation to the surface of a cell so that cell viability and function is preserved.
  • This method to covalently link Ag to the surface of a DC the ability of that cell to function as a competent antigen-presenting cell is preserved.
  • SPDP to function under mild physiological conditions allows for the coupling of a protein antigen to the surface of DCs with no significant reduction in cell viability.
  • heterobifunctional reagents including but not limited to SMPB, SIAB, SMCC, SMPH and SMPT can also be used. Examples of other heterobifunctional reagents can be found in U.S. patent no. 4,529,712 and 4,232,119 (incorporated herein by reference).
  • DCs coupled to Ag and administered as a vaccine or therapy according to the present invention elicit an enhanced immune response compared to the immune response generated by DCs loaded with soluble Ag.
  • covalent linkage of antigen to cell surface proteins of in vitro culture derived DCs will allow for the immunological benefits of receptor mediated Ag loading of DCs without the excessive technological limitations associated with the previous approaches.
  • dendritic cells in which one or more antigens have been covalently linked to surface molecules as described herein.
  • the DCs before and after covalent coupling to Ag can be used fresh or stored frozen.
  • standard method known to those skilled in cell culture techniques can be used. For example, 5 x 10 6 cells/ml in RPMI tissue culture medium containing fetal calf serum (10%) and DMSO (10%) are frozen using a controlled freezing apparatus and stored in liquid nitrogen until they are to be used.
  • the DCs may also be induced to mature in culture. Several factors are known for maturation of DCs including exposure to GM-CSF, LPS or CpG oligonucleotides, or crosslinking of CD40.
  • CpG olignucleotide means an oligonucleotide containing at least one unmethylated CpG dinucleotide.
  • CpG oligonucleotide is as follows: TCCATGACGTTCCTGACGTT (SEQ ID NO:l).
  • Other examples can be found in U.S. patent nos. 6,406,705 and 6,239,116 and in Chu et al., 1997.
  • maturation can be induced in the DCs.
  • a method for making a composition for use in eliciting an immune response comprises the step of obtaining dendritic cells from an individual (or identical twin i.e., syngeneic) and covalently linking the antigen to the surface molecules of the dendritic cells such that the immunogenicity of the antigen is increased over when the Ag is not covalently coupled to the DCs.
  • a method for increasing the immunogenicity of an antigen comprises the steps of obtaining dendritic cells from an individual in need of treatment, covalently linking an antigen to surface protein and glycoprotein molecules of the dendritic cells and reinfusing the dendritic cells into the individual to elicit an immune response. While it is preferable to use dendritic cells of the recipient, dendritic cells from an identical twin (syngeneic) can also be used.
  • the present invention can be used for preventive as well as prophylactic purposes.
  • the following examples are provided to illustrate the present invention and are not meant in any way to be restrictive.
  • N-Succinimydyl-3 (2-pyridyldithio) propionate (SPDP) (Sigma, St Louis, MO) was prepared at 20mM in absolute ethanol and used as outlined below.
  • 2- mercaptoethanesulfonic acid - sodium salt (MESNa) (Sigma, St Louis, MO) is a membrane impermeant reducing agent that was prepared to lOmM in 50 mM Tris, pH 8.6, 100 mM NaCl, 1 mM EDTA, and 0.2% BSA and used as outlined below.
  • Iodoacetamide (Sigma, St Louis, MO) was used at 0.5M or 1M in 0.3M Tris buffer as detailed below.
  • Concanavalin A (Sigma, St Louis, MO) is a tetrameric protein with carbohydrate binding specificity (lectin) that has the ability to induce mitogenic activity in T lymphocytes and to increase the synthesis of cellular products. Con A was used in the ELISPOT assay to provide a positive control for IFN ⁇ production as described below.
  • Lipopolysaccharide (LPS) (Sigma, St Louis, MO) was derived from Escherichia. coli (Serotype 055:B5) and was used as described below.
  • the synthetic oligodeoxynucleotide CpG 1826 was produced by the Biopolymer facility at Roswell Park Cancer Institute and was phosphorothioate- modified to decrease its susceptibility to phosphodiesterase degradation.
  • the sequence of CpG 1826 was obtained from a previous report (Chu et al. 1997).
  • MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide
  • Sigma, St Louis, MO was used as a measure of T cell proliferation.
  • the animals used to isolate DCs were BALB/c mice (Taconic, Germantown, NY) of 8 to 12 weeks of age. In each experiment the animals were age and sex matched. C57BL/6 mice (Taconic, Germantown, NY) were used only as donors for splenocytes that were used in the mixed leukocyte reactions (MLR). C57BL/6 mice (Taconic, Germantown, NY) were also from 8 to 12 weeks of age and were used as a source of responder cells in mixed leukocyte reactions.
  • MLR mixed leukocyte reactions
  • CT26 is an N-nitroso-N-methylurethane induced BALB/c (H-2 d ) undifferentiated colon carcinoma.
  • This tumor grows progressively in BALB/c mice after subcutaneous or intra-venous injection (Wang et al, 1995).
  • the transfection of this tumor with the bacterial lac-Z gene leads to the expression of ⁇ -galactosidase in the tumor cells (Wang et al 1995).
  • This variant of CT26, CT26.CL25 has been established as a progressively growing tumor.
  • CT26.WT and CT26.CL25 were obtained from Dr. Nicholas Restifo (Surgery Branch in the Division of Clinical Sciences, NCI).
  • the P815 cell line is a mouse mastocytoma line of DBA/2 origin (H2 d ).
  • the P815 cell line and the P13.4 cell line that is a beta-galactosidase (beta- gal) expressing P815 subclone were both obtained from Dr. Michael Bevan (University of Washington).
  • CT26.WT and CT26.CL25 were maintained in complete medium; RPMI 1640 (Gibco-BRL, Grand Island NY) supplemented with Penicillin (20 U/ml, Gibco-BRL, Grand Island NY), Streptomycin (20 ⁇ g/ml, Gibco-BRL, Grand Island NY), L-glutamine (2 mM, Gibco-BRL, Grand Island NY), 2-mercaptoethanol (50 ⁇ M, Sigma, St Louis, MO) and 10% heat inactivated and filtered FCS (Gibco-BRL, Grand Island NY).
  • the P815 and P13.4 cell lines were maintained in sterile filtered Dulbecco's Modified Eagle Medium/F12 nutrient mixture with the addition of 10% heat inactivated FCS (Gibco-BRL, Grand Island, NY).
  • Murine bone marrow derived dendritic cells were generated by the method described by Lutz et al. To prepare mouse bone marrow the femurs and tibiae from the desired number of mice were extracted and the surrounding tissue was removed by rubbing with gauze squares. The bones were soaked in 70% ethanol for 2-5 minutes and were then washed with PBS. Both ends of the bones were cut with scissors and the marrow was flushed out with PBS using a 0.45 mm syringe. Clusters within the suspension were dissociated by vigorous pipetting and the cells were washed once in PBS.
  • the bone marrow derived leukocytes were plated in 100mm bacteriological petri dishes at 2x10 cells per dish in 10ml of complete medium supplemented with 200U/ml rmGM-CSF (Peprotech, Rocky Hill, NJ). At day 3, 10ml of complete medium supplemented with 200U/ml rmGM-CSF was added to each of the plates. On day 6 and 8, 10ml of the medium was removed from each plate and was replaced with fresh medium plus 200U/ml rmGM-CSF. DCs were harvested on day 9 for antigen pulse prior to vaccination.
  • FACS staining DCs were collected, counted and their viability was determined by trypan blue exclusion.
  • the cells were subjected to centrifugation at about 250 x g for 3 minutes at 4°C in quench solution (PBS with 1% bovine serum albumin and 0.5% normal rat serum).
  • quench solution PBS with 1% bovine serum albumin and 0.5% normal rat serum.
  • the cells were placed into 4ml culture tubes (Becton Dickinson, Lincoln Park NJ) at 5x10 5 cells per tube, with one tube from each group of cells being set aside as an auto-fluorescence control.
  • the cells were once again subjected to centrifugation at 1500 rpm for 3 minutes at 4°C in 3ml of quench solution.
  • lO ⁇ l blocking immunoglobulin (lmg/ml) was added to each tube with the tubes then being incubated on ice for 10 minutes. Following this incubation, the specific primary labeled, antibodies (Abs) were added at concentrations consistent with the manufacturers instructions. The specific Abs were incubated on ice for 15 minutes. The cells were washed by the addition of 3ml of quench solution for each tube followed by centrifugation at about 250 x g for 3 minutes at 4°C. After this final wash the cells were fixed by the addition of 500 ⁇ l of 3% formalin. The tubes were stored in the dark at 4°C until they were analyzed by flow cytometry.
  • the antibodies used to stain the DCs were; anti-CDl lc (HL3, Armenian hamster IgG, groupl, ⁇ ), anti-CD86 (GL1, rat IgG 2a , K), anti-Ly6c (AL-21, rat IgM, k), and anti-I-A d (M5/114.15.2, rat IgG 2 , K) (BD PharMingen San Diego CA).
  • MLR/MTT assay Spleens were collected from na ⁇ ve C57BL/6 or BALB/c mice. The spleens were disrupted by maceration between two autoclaved frosted microscope slides. The cells were suspended in cold PBS and subjected to centrifugal force (1000 rpm for 7 min.). The cell pellets were then resuspended in ice cold 0.83% ammonium chloride for five minutes. The cells were then spun down (1000 rpm for 7 min.) and washed by resuspending in complete RPMI 1640 medium followed by another centrifugation (1000 rpm for 7 min.). The BALB/c splenocytes were irradiated and used as stimulator cells in the MLR.
  • the C57BL/6 mouse splenocytes were placed into tissue culture flasks at 2xl0 7 cells per flask. After a 1-hour incubation the non- adherent cells were removed and used as responders in the one-way MLR.
  • BALB/c DCs were cultured as described above. The cells were either left unpulsed or pulsed with soluble ⁇ -galactosidase or PDTP- ⁇ -galactosidase.
  • the DCs were irradiated (5000 rad) and then plated in 96 well plates at 2.5x10 4 to 5x10 5 cells per well.
  • BALB/c splenocytes When BALB/c splenocytes were used as stimulator cells they were irradiated (5000 rad) and then plated at lxlO 5 to 5xl0 6 cells per well. The responder cells were added at lxl 0 5 cells per well in a 96 well plate. Control wells were included that contained stimulator cells alone, responder cells alone or medium alone. Each mixture was plated in triplicate wells.
  • MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide
  • Antigen pulse of murine DCs DCs were prepared from mouse bone marrow as described above. The DCs were grown in complete medium. The DCs were harvested on day 9 and washed extensively (4x) in PBS to remove residual medium related protein. The cells were split into three groups for the antigen pulse. Group 1 DCs were pulsed with PBS alone, group 2 DCs were pulsed with soluble ⁇ -galactosidase at lmg/ml PBS for 30 minutes and then lOO ⁇ g/ml complete medium for 16 hours and group 3 DCs were pulsed with PDT- ⁇ -galactosidase at lmg/ml for 30 minutes and then lOO ⁇ g/ml for 16 hours.
  • Each group of cells was initially plated in 2 wells of a 6 well plate at Ixl0 7 /weh7 in 500 ⁇ l PBS or Ag in PBS for 30 minutes. Following the first stage of the Ag pulse 4.5 ml of complete medium, including GM-CSF, was added to each well and the DCs were incubated overnight. At the end of the incubation period 2 ml of medium was removed from each well and replaced with 3 ml of complete medium + 600U GM-CSF and 6 ⁇ g of 1826 CpG. The cells were then incubated for an additional 6 hours. At the end of the final incubation the DCs were collected and washed 4 times in PBS to remove any residual antigen, FCS or CpG. The DCs were counted and injected as indicated in the results section. Preparation of PDTP-proteins
  • ⁇ -Galactosidase was prepared as a 5mg/ml solution in PBS. The protein solution was mixed with 1M iodoacetamide in 0.3M Tris buffer with to bring the solution to 0.022M iodoacetamide. The protein was treated for 30 minutes at room temperature with mixing and then dialyzed against 4 liters of PBS at 4°C for 16 hours. At this point half of the protein was set aside for use as the source of soluble ⁇ -galactosidase.
  • the protein solution was prepared in 2ml of PBS at a concentration of 2.5 mg/ml, 20mM SPDP was prepared in absolute ethanol and 53.8 ⁇ l was added to the 2ml of protein solution. The reaction was carried out for 30 minutes at room temperature with mixing. The solution was then dialyzed against 4 liters of PBS at 4°C for at least 16 hours. Protein concentration for both ⁇ -galactosidase and PDTP- ⁇ -galactosidase was determined by optical density read at 280nm. The preparations were diluted to lmg/ml of protein in PBS and sterilized using a 0.22 ⁇ M filter prior to use as antigen. The protein was then used or apportioned and frozen.
  • dendritic cells were incubated with PBS, soluble ⁇ -galactosidase in PBS or PDTP- ⁇ - galactosidase for 30 minutes at 4°C. The cells were then washed three times in PBS+5% BSA. After the final wash the cells were resuspended in PBS. An equal amount of the developing substrate containing o-nitrophenyl- ⁇ -Z)-galactopyranoside (ONPG) was added to lOOul of cell suspension in PBS. The cells were incubated for 5 minutes at 37°C in a round bottom 96 well plate. After the incubation the plate was centrifuged for 5min at 2000 rpm at 4°C to pellet the cells. After the centrifugation 150ul of the supernatant was transferred to a flat bottom 96 well plate that was read at 450nm.
  • PBS soluble ⁇ -galactosidase in PBS or PDTP- ⁇ - galactosidase for 30 minutes at
  • DCs were incubated with PDTP- ⁇ -galactosidase for 30 minutes on ice. The cells were then incubated at 37°C in a water bath for a given length of time. At the end of each incubation period the cells were removed from the water bath and placed on ice. When all of the incubation time points were reached the cells were washed three times with PBS + 5% BSA. Centrifugation was carried out at 1000 rpm for 7 minutes at 4°C.
  • the cell pellets were resuspended in a solution comprised of 50 ⁇ l of lOmM mercaptoethanesulfonic acid (MESNa) in 50 mM Tris, pH 8.6, 100 mM NaCl, 1 mM EDTA, and 0.2% BSA.
  • MESNa lOmM mercaptoethanesulfonic acid
  • the cells were reacted for 30 minutes at 4°C with gentle mixing.
  • a second amount of MESNa was added (12.5 ⁇ l of a 50mM stock, freshly prepared before addition) and the cells were incubated at 4°C for 30 minutes.
  • MESNa 16ul of a 50 mM stock
  • Spleens were collected from vaccinated or na ⁇ ve mice. The spleens were disrupted by maceration between two autoclaved frosted microscope slides. The cells were suspended in cold PBS and centrifuged (1000 rpm for 7 min.). The cell pellets were then resuspended in ice cold 0.83% ammonium chloride for five minutes. The cells were spun down (1000 rpm for 7 min.) and resuspended in complete RPMI 1640 medium followed by another centrifugation (1000 rpm for 7 min.). The supernatant was removed and the cells were resuspended to 2xl0 6 cells per milliliter in 5 ml complete RPMI 1640 medium.
  • CD8 + cell separation At the end of the restimulation period the cultured cells were collected from the culture dishes and were washed by centrifugation (1000 rpm for 7 min.). The cells were counted and portion of the cells was set aside for use in the ELISPOT assay; these cells were termed unfractionated. The second portion of the cells from each group was prepared for CD8 + magnetic bead enrichment using the MACS microbeads system (Miltenyi Biotech, Aubern CA) according to the company's protocol. These cells were washed in cold MACS buffer (PBS, 2mM EDTA and 0.5% BSA). The cell pellet was resuspended in 90 ⁇ l of buffer per 10 7 total cells.
  • MACS microbeads system MACS microbeads system
  • the plate was carefully checked to be sure that no air was trapped in the wells during the washes.
  • the solution was removed from the wells by pouring out and flicking over a sink followed by tapping on clean paper towels.
  • Responder cells were recovered from the restimulation cultures and were added at varying concentration to the wells in lOO ⁇ l volumes of complete medium (RPMI 1640 + 10% FCS).
  • CD8 + cells were separated from the remainder of the cultured cells (as above). Typically the cells were assayed in four replicates.
  • Target cells, in lOO ⁇ l of complete medium were then added to the wells.
  • the target cells can consist of PI 3.4 and CT26.CL25 tumor cells, CT26.WT cells, and P815 tumor cells, with or without peptide.
  • Splenocytes from each group being tested were also plated in lOO ⁇ l of complete medium plus lOO ⁇ l of 20ug/ml concanavalin A (Sigma, St Louis, MO) and serve as positive controls for the release of IFN ⁇ .
  • the plate was then incubated overnight at 37°C in 5% CO 2 . Following the overnight incubation, the wells were emptied as above and washed 6 times with PBS containing 0.05% Tween 20, with each wash being incubated for 3 minutes.
  • the detection Ab was a biotinylated anti-mouse IFN ⁇ Ab (clone XMGl .2), 0.5 ⁇ g/ml in 50 ⁇ l was added to each well. The wells were then incubated at 37°C in 5% CO 2 for 2 hours. Following this incubation the wells were emptied and washed 6 times as described above. Avidin-horseradish peroxidase complex (Vectastain Elite Kit, Vector Scientific, Burlingame, CA) was prepared in PBS with 0.1% Tween 20. Each well receives lOO ⁇ l of the Vectastain solution and the plate was incubated for 1 hour at room temperature.
  • the peroxidase substrate solution was prepared by first dissolving one tablet of 3-amino-9- ethylcarbazole (AEC; Sigma, St Louis, MO) in 2.5ml dimethylformamide (DMF). This solution was then added to 47.5 ml of 50mM acetate buffer. Immediately before use, 25 ⁇ l of 30% hydrogen peroxide was added and the solution was filtered to remove any particulate material that did not go into solution.
  • the reaction was stopped by briefly washing the plates with cold tap water. The water was emptied from the plate and the plate was blotted on clean paper towels. The plastic bottom was removed from the plates, which were then left to dry overnight at room temperature. The next day the plates were counted by eye with the aid of a dissecting microscope.
  • lOO ⁇ l of anti-mouse IL-12 p70 conjugate (horseradish peroxidase labeled secondary Ab).
  • the plate was mixed and incubated for 2 hours at room temperature.
  • the plate was washed four more times and lOO ⁇ l of substrate solution (tetramethylbenzidine and hydrogen peroxide) was added to the wells.
  • the plate was incubated for 30 minutes in the dark at room temperature.
  • the lOO ⁇ l of stop solution was added to each well and the optical density of the individual wells was determined by reading the plates in a microplate reader set to 450nm with a correction reading taken at 540nm.
  • the total concentration of IL-12 present in the supematants was determined by plotting the OD values against a standard curve.
  • the Flow Cytometry department at Roswell Park Cancer institute performed a mouse cytokine array on tissue culture supematants from DC cultures.
  • the flow cytometer microsphere based assay allows for the simultaneous detection of multiple soluble cytokines in one sample tube (Reviewed in Vignali, 2000).
  • the mouse cytokines that were tested were: IL-1 ⁇ , IL-6, and TNF ⁇ .
  • mice In vivo vaccination studies Groups of BALB/c mice were injected with unpulsed DCs, soluble ⁇ -gal pulsed DCs, PDTP- ⁇ -gal DCs or PBS. All of the DCs were exposed to 1 ⁇ g/ml CpG 1826 to induce maturation. A minimum of seventeen days after immunization the mice were challenged with a subcutaneous injection of ⁇ -gal expressing CT26.CL25 tumor cells or the parental CT26.WT tumor cells. The tumors were measured weekly to determine growth rate and the mice were monitored closely for signs of morbidity. Mice were sacrificed when any dimension of their tumor reached or exceeded 2 cm in size.
  • Dendritic cell biology phenotype, maturation, and function GM-CSF cultured Bone marrow cells have the morphological characteristics of dendritic cells.
  • One of the hallmarks of a dendritic cell is its unique morphology.
  • Steinman In the early seventies described cells that had an unusual dendritic shape with continually forming and retracting processes (Steinman and Conn 1973, Steinman and Cohn 1974).
  • the technology to generate DCs from bone marrow was perfected the cells obtained were described similarly as possessing a distinct dendritic cell shape with sheet like processes or veils (friaba et al. 1992).
  • bone marrow cells were cultured in complete medium containing 200U/ml rmGM-CSF as described in the methods section. On day 10 of the culture the cells were removed, washed, and placed on a lysine coated glass slide. The cells were then examined microscopically. As shown in figure 1, the cells obtained from GM-CSF stimulated bone marrow cell cultures exhibit the "veiled" dendritic cell morphology with the attached cells having the stellate shape that is typical of DCs plated in this manner (Sallusto et al. 1995).
  • GM-CSF cultured Bone marrow cells have a DC phenotype as determined by FACS analysis.
  • the phenotype of bone marrow derived DCs can be monitored through the use of flow cytometry. Cells recovered from the cultures on day 7 were washed and subjected to analysis by flow cytometry. By side scatter (SSC) and forward scatter (FSC) analysis the non-adherent fraction of the cultured cells displayed the low granularity (SSC) and variable cell size (FSC) typical of bone marrow derived DCs (Lutz et al. 1999) (figure 2a). Phenotypic markers characteristic of immature DCs were also examined. The expression of moderate levels of CDllc (figure 2b) and MHC class II antigens (figure 2c) found by immunophenotyping are indicative of immature DCs (Inaba et al. 1992, Lutz et al. 1999).
  • bone marrow derived DCs Another defining feature of bone marrow derived DCs is a well-defined cellular response to a maturation signal.
  • bone marrow derived DCs upregulate surface expression of costimulatory molecules. This upregulation can be quite marked, with the surface expression levels of the costimulatory molecule CD86 increasing up to 100 fold (Mellman and Steinman 2001). Maturation is also accompanied by an increase in the expression level of MHC class II. Flow cytometric analysis of these surface molecules allows for verification of the maturation state of a DC. Two molecules that are known to induce maturation of DCs were used.
  • lipo- polysaccharide LPS
  • a bacterial DNAderived, immunostimulatory oligonucleotide that contains unmethylated CpG repeats are both known to induce DC maturation (Roake et al. 1995, Jakob et al. 1998).
  • the BM cells were first cultured for 9 days, as outlined above. On the ninth day the cells were collected and replated in a six well culture dish. Cells in separate wells received complete medium containing LPS (lOOng/ml), CpG 1826 (1 ⁇ g/ml or 6 ⁇ g/ml) or complete medium alone. Sixteen hours later the cells were recovered, washed, labeled and assayed by flow cytometry for expression of MHC class II and CD86. In accordance with earlier findings (Inaba et al. 1992, Lutz et al. 1999) MHC class II and CD86 expression was increased in these cells by addition of LPS or CpG (figure 3).
  • AUogeneic T cells from C57B1/6 mice were added at 1 x 10 5 cells per well and were mixed with varying doses of irradiated treated BM -DCs or splenoctyes. After a three-day incubation period, cell activity (cell number equivalent) was determined by an MTT assay (Mossman 1983, Maghni et al. 1999) as outlined in the methods section. In these experiments the BM-DCs were found to be at least 50 times more efficient than BALB/c splenocytes in stimulating an MLR (figure 4). These results were repeated twice more with essentially the same results, showing that the BM-DCs were indeed potent stimulators of an MLR.
  • Bone marrow derived DCs produce IL-12 in response to bacterial DNA oligonucleotides that contain unmethylatedCpG motifs. Potent stimulation of T cells in an MLR is considered the most convenient assay for demonstrating the ability of mature DCs (Inaba et al. 1987, Boog et al. 1988) to stimulate T cell proliferation. Yet another, very important aspect of DC activity is the delivery of a so-called third signal (Kalinski et al. 1999).
  • a DC supplies three signals to a na ⁇ ve T lymphocyte. The first signal gives antigenic information by means of a peptide in the context of an MHC molecule. The second signal provides costimulation that is a gauge of the "danger" concomitant with that antigen.
  • the third signal provides information on the type of antigen present and directs the polarization (T H I VS. T H 2) of the primary T- cell response.
  • the MLR allows for the assessment of the first two signals; allogeneic MHC provides the first signal and the costimulatory molecules present on the DC provide signal 2.
  • the potential for delivering signal three can be measured by monitoring DC production of the T H I biasing cytokine IL-12. Production of the p70 heterodimer IL-12 by DCs is clearly correlated with sensitization of T H I lymphocytes in vitro and in vivo (Hilkens et al. 1997, Trinchieri, 1998).
  • GM-CSF BM culture cells were split into 5 groups and transferred to a tissue culture treated 12 well plate. The cells were transferred in 500 ⁇ l of complete medium with 200U/ml GM-CSF at a concentration of 5x10 5 cells/well to duplicate wells. The first group received no additional factors.
  • the remaining groups were treated with 500 ⁇ l medium containing LPS (200ng/ml), or varying doses of CpG-1826 (Chu et al. 1997), viz. 2 ⁇ g/ml, 12 ⁇ g/ml, or 24 ⁇ g/ml.
  • the cells were incubated overnight at 37°C with 5% CO , and then the supernatant fluids were assayed for the production of IL- 12 by ELISA. Neither transfer of the cells to medium alone nor transfer of the cells to medium containing LPS induced IL-12 production (figure 5). Increasing amounts of CpG did not cause the production of greater amounts of IL-12 (p70).
  • Previous reports have shown that the optimal concentration of CpG used to elicit IL-12 production in vaccines and a DC- cell line culture was 6ug/ml. In my experiments using concentrations greater than
  • CpG 1826 Using the dose of 1 ⁇ g/ml CpG 1826 will not only allow for maximal IL-12 release by these cells but will also induce their maturation as seen by the upregulation of MHC II and CD86 displayed in figure 3.
  • the experiments using the CpG-1826 establish that the cells derived from the 9 day in vitro culture in GM-CSF are able to respond to bacterial DNA in a manner that is consistent with their characterization as dendritic cells and consistent with their ability to provide a T H I biased third signal.
  • the cells described here are morphologically, phenotypically, and functionally dendritic cells.
  • the cells possess the morphological characteristics and the CD1 lc and MHC class II expression that is typical of DCs. Also consistent with their identity as DCs, the cultured cells upregulate the costimulatory molecule CD86 and increase the levels of MHC class II molecules on their cell surface upon stimulation with maturation factors like LPS and CpG. A high level of T-cell stimulation in the allogeneic MLR confirms that these cells possess the functional, and immunostimulatory capabilities of DCs.
  • the cultured cells when cultured in the presence of bacterial CpG oligonucleotides, the cultured cells have the ability to produce IL- 12, a cytokine that is crucial to the elicitation of a T H I response to a given antigen.
  • the ability to culture and isolate functional DCs provides an opportunity to test the efficacy of the covalent linkage of Ag to proteins on the surface of DCs as a vaccine for cancer immunotherapy and to compare this novel strategy of DC Ag loading to conventional Ag loading protocols.
  • ⁇ -galactosidase was used as a model tumor specific antigen.
  • ⁇ -galactosidase and its modification by SPDP ⁇ -Galactosidase is a bacterial enzyme that is known to induce a T cell mediated immune response when presented by DCs and has been used as a surrogate tumor antigen in investigations into the treatment of cancer in laboratory animals (Wang et al 1995, Irvine et al 1996, Paglia et al. 1996, Specht et al. 1997, and Brunner et al 2000).
  • Vaccinating mice with soluble ⁇ -gal pulsed murine BMDCs evokes a protective anti-tumor response in 40% of the vaccinated mice (Paglia et al. 1996,).
  • Heterobifunctional reagents such as SPDP, link to the protein in the first reaction and then link to the surface membrane protein in the second reaction. These reactions are performed in separate sequential steps, in a process that allows the two reactive groups of SPDP to react with two different targeted functional groups and thereby avoids cross- linking of antigen molecules (figure 7).
  • PDTP modified proteins should bind to the surface proteins of a cell through the formation of a covalent disulfide bond. If this occurs then cleavage of the newly formed disulfide bond would liberate ⁇ -gal from the cell surface and lead to a reduction in the ⁇ -gal activity observed on the surface of the DCs.
  • Mercaptoethanesulfonic acid sodium salt (MESNa) was used to selectively cleave disulfide bonds present in proteins on the surface of the DCs.
  • PDTP- ⁇ -gal loaded DCs were incubated at 37°C for 0 and 30 minutes and the surface ⁇ -gal cleaved with MESNa. The DCs were split into two different groups at each time point. The cells were then washed thoroughly. Half of the cells from each of the 0 and 30 minute incubation time points were subjected to freeze thaw lysis and half were left as intact cells. The cells or an equal amount of cell equivalents in lysis supernatant was then assayed for ⁇ -gal activity.
  • Figure 10 shows that a significant amount of ⁇ - gal was protected from cleavage with MESNa as it was found in the cell lysate after a 30 minute incubation at 37°C. This is consistent with the theory that a significant portion of the enzyme linked to the surface was internalized constitutively just 30 minutes after the initiation of a 37°C incubation period. No evidence of intemalization was observed during a 30-minute incubation period at 4°C (data not shown). It can be seen that antigen can be covalently linked to the surface proteins of DCs and those cells can internalize that antigen.
  • the ⁇ -gal that is loaded on DCs through covalent linkage is believed to be degraded after intemalization. Proteins degraded by APCs such as DCs are presented as peptides associated with surface MHC molecules.
  • Covalent coupling of ⁇ -Gal to DCs does not alter the cell's functional ability to stimulate na ⁇ ve T cells in an MLR. It was important to determine whether the covalent linkage of ⁇ -gal to the surface of the DC would change the cell's stimulatory capacity in a one-way MLR. To this end DCs were cultured overnight in the absence of Ag, with soluble ⁇ -gal or with PDTP ⁇ -gal. After washing away unbound Ag, increasing numbers of irradiated DCs were mixed with C57B1/6 mouse lymphocytes.
  • DCs were first pulsed overnight with soluble ⁇ -gal or PDTP- ⁇ -gal or left unpulsed. Medium that was added the next day was standard medium that contained CpG 1826 (1 ⁇ g/ml).
  • a flow cytometric bead ELISA was used to determine the amounts of IL-l ⁇ , IL-6, and TNF ⁇ released from unpulsed DCs, soluble ⁇ -gal pulsed DCs and ⁇ -gal-conjugated DCs. Table 1 shows the results of the cytometric bead ELISA.
  • Colon 26 is a N-nitroso-N-methylurethane induced colon carcinoma established in a BALB/c mouse. This tumor is poorly immunogenic and grows progressively in animals after subcutaneous or intravenous injection (Wang 1995). The tumor has been transfected with the bacterial lac-Z gene, which causes the cells to express ⁇ - galactosidase that serves as an experimental tumor antigen. Because there are many assays to detect the presence and activity of ⁇ -gal, the ease of detection of this model tumor antigen has made it a popular choice for immunological investigations. Pulsing of DCs with the soluble form of the model tumor Ag ⁇ -gal is an established practice in experimental immunotherapy.
  • PDTP- ⁇ -galactosidase pulsed DC vaccination is superior to soluble ⁇ -galactosidase pulsed DC vaccination in protecting mice from challenge with a ⁇ -gal expressing tumor.
  • the first step in establishing the efficacy of an anti-tumor vaccine is to establish its ability to induce protective immunity in vivo. Accordingly, groups of BALB/c mice were injected with unpulsed DCs, soluble ⁇ -gal pulsed DCs, PDTP- ⁇ - gal DCs or PBS. All of the DCs were exposed to 1 ⁇ g/ml CpG 1826 to induce maturation. Seventeen days after a single immunization the mice were challenged with a subcutaneous injection of ⁇ -gal expressing CT26.CL25 tumor cells.
  • mice vaccinated with unpulsed DCs were attained in 13.3% of mice vaccinated with unpulsed DCs, 43.3% of soluble ⁇ -gal pulsed DC vaccine recipients, and 90.0% of PDTP- ⁇ -gal pulsed DC vaccinated animals.
  • the results presented in figure 12 reveal a highly significant advantage to vaccination with antigen conjugated DCs when compared to DCs co-incubated with soluble ⁇ -gal (p ⁇ O.00011).
  • Tlie anti-tumor protection provided by DC vaccination is Ag specific. Vaccinating mice with DCs, and then challenging them with ⁇ -gal negative CT26.WT cells addressed the issue of specificity. The vaccinations were carried out using the established protocol of 5x10 5 DCs per mouse. The four vaccination groups consisted of unvaccinated mice or vaccination with unpulsed DCs, soluble ⁇ -gal pulsed DCs, or PDTP- ⁇ -gal pulsed DCs. The growth of non ⁇ -gal expressing CT26.WT tumors was progressive in all four groups challenged with that parental cell line (figure 13). One mouse that was vaccinated with soluble ⁇ -gal pulsed DCs did not demonstrate any tumor growth.
  • the DCs prepared for this experiment were capable of protecting mice from challenge with the ⁇ -gal expressing CT26.CL25 tumor cells.
  • PDTP- ⁇ -gal pulsed DCs provided protection from the ⁇ -gal expressing CT26.CL25 tumor challenge.
  • the immune response to soluble ⁇ -gal pulsed and ⁇ -gal-conjugated DCs is specific, as vaccination with these preparations only protect against challenge of a tumor that expresses ⁇ -gal.
  • the specificity exhibited here is one of the hallmarks of an adaptive immune response.
  • Another measure of the adaptive immune response is memory.
  • PDTP- ⁇ -galactosidase pulsed DCs but not DCs pulsed with soluble ⁇ -gal suppress or eliminate established ⁇ -gal positive tumors.
  • a therapeutic model was established to test the efficacy of DC vaccine in a more clinically relevant setting. Mice were inoculated with 5xl0 5 CT26.CL25 cells subcutaneously on their left flank. The tumors were allowed to establish and grow for 10 days at which point the mice were redistributed into the three groups that would receive one of three different DC therapies. The tumor sizes were measured and documented on day 10.
  • mice receiving soluble ⁇ -gal pulsed DCs had an average tumor size of 52.30 mm 3 (21 to 90 mm 3 range), and the third group of animals with an average tumor size of 52.82 mm (26 to 104 mm range) received DCs with covalently coupled ⁇ -gal.
  • the DCs were prepared as in previous experiments, including the addition of 1 ⁇ g/ml CpG in the final 6 hours of the in vitro culture. The DCs were washed thoroughly and injected s.c. on the flank opposite the established tumor, i.e. on the right side. The mice were monitored closely and tumor measurements were taken weekly.
  • mice receiving unpulsed DCs and soluble ⁇ -gal pulsed DCs did not respond to the treatment, as evidenced by the continued growth of the established CT26.CL25 ( ⁇ -gal expressing) tumors (figure 15).
  • CT26.CL25 ⁇ -gal expressing tumors
  • mice treated with ⁇ -gal conjugated DCs evidence of tumor suppression or complete eradication was observed.
  • mice treated with PDTP- ⁇ -gal loaded DCs rapid involution of the s.c. tumors was observed.
  • Such a rapid anti-tumor effect is suggestive of an innate immune response.
  • a clear advantage can be seen in the covalent coupling of antigen to DCs when compared to a soluble pulse of DCs with that same Ag.
  • DCs require a maturation stimulus to evoke a response that can be detected in an ELISPOT assay.
  • the DCs used in the vaccinations were exposed to CpG 1826 prior to their injection into mice.
  • Four groups of DCs were prepared, two plates each of unpulsed DCs and soluble ⁇ -gal pulsed DCs. After an overnight DC incubation period with or without ⁇ -gal, one plate of unpulsed DCs and one plate of ⁇ -gal pulsed DCs were treated with 1 ⁇ g/ml of CpG for 6 hours as a maturation stimulus.
  • mice Twelve days after the vaccination the mice were sacrificed and their splenocytes were restimulated in vitro for 6 days by co-culturing them with irradiated ⁇ -gal expressing P13.4 tumor cells at a ratio of 100 splenocytes to 1 tumor cell.
  • the PI 3.4 cell line is a subclone of the DBA/2 (H-2d) mastocytoma P815 that expresses ⁇ -galactosidase (Carbone and Bevan 1990).
  • the cells from the four groups were collected and distributed in two fold dilutions to an ELISPOT plate that had been coated with an anti-TFN ⁇ antibody. The cells were then cultured overnight with the addition of the ⁇ -gal expressing
  • PDTP- ⁇ -gal pulsed DCs elicit a ⁇ -gal specific response that is equal to the response seen with soluble pulsed ⁇ -gal DCs.
  • An in vitro analysis of a the immune response generated by ⁇ -gal conjugated DC vaccination and soluble ⁇ -gal pulsed DC vaccination was undertaken to compare the ability of the two protocols to elicit IFN ⁇ production in T cells.
  • Mice were vaccinated intraperitoneally with 5x10 5 unpulsed DCs, soluble ⁇ -gal or PDTP- ⁇ -gal pulsed DCs (all were matured using CpG 1826).
  • the splenocyte collection and 6 day in vitro restimulation (IVS) was carried out as previously stated with the stimulator cells being irradiated PI 3.4 cells.
  • ⁇ -Gal is required for IFN ⁇ production in the ELISPOT assay.
  • mice were vaccinated with unpulsed DCs, DCs pulsed with soluble ⁇ -gal or DCs pulsed with PDTP- ⁇ -gal.
  • the splenocytes from each vaccinated group were split into two groups for restimulation upon recovery. The first restimulation was with co-cultured irradiated, ⁇ -gal negative CT26.WT tumor cells and the second restimulation group was co- cultured with ⁇ -gal expressing CT26.CL25 cells.
  • in vitro assays such as the ELISPOT and CTL assays do not correlate with the outcome of the overall immune response in vivo (Dallal and Lotze 2000).
  • the production of IFN ⁇ as measured by the ELISPOT assay is only one parameter that can be used as an in vitro measure of a vaccine's efficacy. Therefore a more telling measure of vaccine efficacy can be addressed in murine models by measuring immune activity in vivo, using protection from or treatment of a tumor challenge.
  • the measurement of IFN ⁇ production by restimulated splenocytes may be too narrow a parameter to measure the efficacy of a protective or therapeutic anti-cancer vaccine.
  • Ag covalently linked to the surface of DCs has been found to be superior to that of DCs pulsed with soluble Ag in the induction of protective and therapeutic anti-tumor immunity.

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Abstract

L'invention concerne une méthode permettant de renforcer l'immunogénicité d'un antigène. Cette méthode implique, d'une part, la liaison covalente de l'antigène à des protéines ou à des glycoprotéines présentes à la surface de cellules dendritiques au moyen d'une légère modification biochimique qui minimise la dénaturation de l'antigène et, d'autre part, la préservation de la viabilité cellulaire. Les cellules dendritiques comportant, sur leur surface, un antigène lié par covalence peuvent servir à générer une réponse spécifique à l'antigène. Cette méthode peut servir à des fins thérapeutiques ou prophylactiques.
EP02796072A 2001-12-18 2002-12-18 Methode permettant de renforcer l'immunogenicite par liaison covalente d'antigenes a des proteines a la surface de cellules dendritiques Withdrawn EP1465657A4 (fr)

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